Particulate pharmaceutical composition

ABSTRACT

The present invention provides a particulate pharmaceutical composition which has improved drug encapsulation stability and is suitable for a drug delivery system. The particulate pharmaceutical composition  1  contains: a plurality of block copolymer unit  2  arranged radially, each of which has a hydrophobic polymer-chain segment  2   b , which is arranged radially inside, and a hydrophilic polymer-chain segment  2   a , which is arranged radially outside; a drug  4 , which includes a biomacromolecule; and a charged lipid  3 , which has an electrical charge opposite to that of the drug  4 ; wherein the charged lipid  3  is being attracted to the hydrophobic polymer-chain segment  2   b , and the drug  3  is positioned radially inside the hydrophobic polymer-chain segment  2   b . The pharmaceutical composition  1  can effectively prevent the drug  4  from disengaging from the particle.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Phase Patent Application and claims thepriority of International Application No. PCT/JP2011/053052, filed onFeb. 14, 2011, which claims priority of Japanese Patent Application No.2010-029486, filed on Feb. 12, 2010.

INCORPORATED BY REFERENCE

The material in the text file entitled “Y828.txt,” amended Apr. 4, 2011and being 1,880 bytes in size, is herein incorporated by reference inits entirety.

TECHNICAL FIELD

The present invention relates to a particulate pharmaceuticalcomposition that can be used as a drug delivery system (DDS) and isconstituted of a drug and a particulate carrier compositionencapsulating the drug.

BACKGROUND ART

Biotechnology-based pharmaceuticals, which utilize biomacromoleculessuch as proteins and nucleic acids, are more susceptible to enzymaticdegradation or immune elimination, compared with conventionalpharmaceuticals based on low-molecular compounds. Patent Documents 1 to3 disclose a DDS which contains a biomacromolecule within a liposomemade of a lipid bilayer membrane, which intend to improve the in vivostability of biotechnology-based pharmaceuticals.

PRIOR ART REFERENCES Patent Documents

Patent Document 1: WO2001/034115

Patent Document 2: WO1998/58630

Patent Document 3: WO2005/092389

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The conventional DDSs described in Patent Documents 1 to 3, in which thebiomacromolecule drug is protected with a lipid bilayer membrane, aresuperior in in vivo stability of the drug, but are inferior in drugreleasability from the carrier. In addition, due to the large particlesize and also due to the electrical charge of the lipid whichconstitutes the lipid bilayer membrane, the conventional DDSs are likelyto be captured by the reticuloendothelial system, such as the lungs,liver and spleen, and thereby removed from blood before reaching to theadministration target.

A polymeric micelle formed with a block copolymer unit having ahydrophobic polymer-chain segment and a hydrophilic polymer-chainsegment can be used as a DDS carrier, and the resultant DDS can be muchsmaller in particle size (e.g., the average particle size can be 100 nmor smaller) than the conventional DDSs using a liposome. However, such aDDS using a polymeric micelle as the carrier still has difficulty, insome cases, in delivering the drug to the administration target, due tolack of sufficient encapsulation force to maintain the biomacromoleculewithin the DDS particle as shown in the Comparative Examples, which willbe explained later. In addition, such a DDS may sometimes cause the drugto disengage from the carrier during the storage period afterproduction.

The present inventors have developed a polymeric micelle DDS. The outersurface of the micelle DDS is prevented from gathering a chargedsubstance as a corollary of less electrical charge (Japanese PatentApplication No. 2009-200681). This DDS is prevented from mis-deliveringof drugs to the administration target as a corollary of less adhesion ofa biomolecule onto the carrier surface. However, this DDS still has roomto extend a duration of the drug encapsulation effect.

Means to Solve the Problems

The present invention provides a particulate pharmaceutical compositioncontaining a block copolymer unit having a hydrophobic polymer-chainsegment and a hydrophilic polymer-chain segment; a drug; and a chargedlipid carrying a charge opposite to the charge of the drug. The drugincludes at least a biomacromolecule selected from the group consistingof a protein and a nucleic acid. In the particulate pharmaceuticalcomposition, a plurality of the block copolymer units are arrangedradially with the hydrophobic polymer-chain segments radially inside andthe hydrophilic polymer-chain segments radially outside. The chargedlipid is being attracted to the hydrophobic polymer-chain segment. Thedrug is positioned radially inside the hydrophobic polymer-chainsegments, whereby the drug is prevented from disengaging from theparticle.

Effects of the Invention

The pharmaceutical composition according to the present invention hasimproved drug encapsulation stability and is suitable for DDS. Thispharmaceutical composition can deliver the drug more reliably than theconventional DDSs, and is especially useful for an administration targetthat requires a longer period of drug delivering.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1( a) and 1(b) illustrate an example of the structure of thepharmaceutical composition of the present invention;

FIGS. 2( a) and 2(b) illustrate an example of drug distribution changein the particle between before and after freezing operation;

FIGS. 3( a) and 3(b) indicate evaluation results of drug encapsulationstability of pharmaceutical compositions; and

FIGS. 4( a) and 4(b) indicate evaluation results of blood circulation ofpharmaceutical compositions.

DESCRIPTION OF EMBODIMENTS

FIGS. 1 and 2 are referred to in the following description only for thepurpose of helping the understanding of the present invention. FIGS. 1and 2 are mere illustrative diagrams to which the present inventionshould not be limited. For example, although FIGS. 1 and 2 illustrate anexample in which the charged lipid is cationic and the drug is anionic,the present invention should not be limited to this example.

FIG. 1 (a) illustrates an example of the structure of the particulatepharmaceutical composition according to the present invention(hereinafter also referred to as “pharmaceutical composition”). Thepharmaceutical composition 1 contains a block copolymer unit 2, acharged lipid 3, and a drug 4. FIG. 1 (b) is an enlarged view of theblock copolymer unit 2, which has a hydrophilic polymer-chain segment 2a and a hydrophobic polymer-chain segment 2 b. The block copolymer units2 are arranged radially in the pharmaceutical composition 1 with thehydrophobic polymer-chain segments 2 b radially inside and thehydrophilic polymer-chain segments 2 a radially outside. The chargedlipid 3 carries a charge opposite to the charge of the drug 4, and isbeing attracted to the hydrophobic polymer-chain segments 2 b.

In the pharmaceutical composition 1 according to the present invention,the drug 4 is positioned radially inside the hydrophobic polymer-chainsegments 2 b, as shown in FIG. 1 (a). This does not mean that all of thedrugs 4 contained in the pharmaceutical composition 1 must be positionedradially inside the hydrophobic polymer-chain segments 2 b; some of thedrugs 4 may be positioned radially outside the hydrophobic polymer-chainsegments 2 b. This arrangement in the pharmaceutical composition 1 ofthe present invention serve to prevent the drugs 4 from disengaging fromthe particle, i.e., to improve encapsulation stability of the drugs 4.

The pharmaceutical composition 1 can be produced by, e.g., carrying outa freezing operation on a pharmaceutical composition precursor, in whichthe drug is positioned outside the hydrophobic polymer-chain segments 2b. The pharmaceutical composition precursor 1′ can readily be formed byincorporating the drugs into a carrier composition in a known manner, aswill be explained later. FIG. 2( a) illustrates distribution of thedrugs 4 in the particle of the pharmaceutical composition precursor 1′before the freezing operation, and FIG. 2( b) illustrates distributionof the drugs 4 in the particle of the pharmaceutical compositionprecursor 1′ after the freezing operation. As shown in FIG. 2( a), thedrugs 4 are positioned outside the hydrophobic polymer-chain segments 2b in the particle of the pharmaceutical composition precursor 1′.Through the freezing operation, the drugs 4 move radially inward,resulting in the pharmaceutical composition 1 in which, as shown in FIG.2( b), the drugs 4 are positioned radially inside the hydrophobicpolymer-chain segments 2 b. Thus, the pharmaceutical composition 1 ofthe present invention can be produced by transferring the drugs 4, whichare positioned radially outside the hydrophobic polymer-chain segments 2b in the pharmaceutical composition precursor 1′, to radially inside thehydrophobic polymer-chain segments 2 b via the freezing operation. Thereason why such drug transfer occurs is not exactly clear, but isbelieved that the arrangement of the block copolymers 2 and the chargedlipids 3 forming the carrier composition is disturbed by the freezingoperation to cause gaps, through which the drugs 4 are introduced intothe inner part of the particle. The freezing operation may be carriedout at least once, but should preferably be carried out twice or more.Repeating the freezing operation can facilitate introduction of thedrugs 4 into the inner part of the particle.

The freezing operation may be any operation as long as it involvesfreezing of a certain composition, such as a freeze-drying operation anda freezing-and-thawing operation.

The freeze-drying (lyophilyzation) operation includes the steps of:freezing the composition (freezing step A); and drying the frozencomposition (drying step). Freezing step A can be performed bymaintaining the composition at a temperature of −200° C. or higher,preferably −100° C. or higher, and −10° C. or lower, preferably −20° C.or lower for a period of an hour or longer, preferably 5 hours orlonger, and 72 hours or shorter, preferably 24 hours or shorter. Dryingstep can be performed by depressurizing the ambient pressure of thefrozen composition to a vacuum state (e.g., 15 Pa or lower) to inducethe water content to sublime. In order to facilitate sublimation, theambient temperature should preferably be raised during depressurizing,either stepwise or continuously, to a temperature higher than thetemperature at the freezing step, e.g., −20° C. or higher or −10° C. orhigher. The upper limit for the raised ambient temperature may be about25° C. The time duration of the drying step may be 5 hours or longer,preferably 20 hours or longer. The upper limit for the time length ofthe drying step may be, although not limited thereto, 100 hours. Sincethe pharmaceutical composition 1 obtained through the freeze-dryingoperation is in the dry state, it should preferably be dissolved into aknown solvent, such as water when used.

Freezing-and-thawing operation includes the steps of: freezing thecomposition in a similar manner to the freezing step A; and thawing thefrozen composition (thawing step). Thawing step can be performed bymaintaining the composition at a temperature of 4° C. or higher,preferably 10° C. or higher, and 40° C. or lower, preferably 30° C. orlower for a period of 30 minutes or longer, preferably an hour orlonger, and 24 hours or shorter, preferably 5 hours or shorter.

Whether the drugs 4 are positioned radially inside the hydrophobicpolymer-chain segments 2 b can be determined based on, e.g., whether theabsolute value of the zeta potential of the pharmaceutical composition 1is higher than that of a drug-containing particle which has the sameconstitution as the pharmaceutical composition 1 but is produced withoutfreezing operation. This is because the drugs 4 move away from the outersurface of the particle toward the inner part of the particle throughthe freezing operation, whereby the charged lipids 3 increase itsinfluence on the absolute value of the zeta potential of thepharmaceutical composition 1.

The charged lipid 3 herein means either an anionic lipid, which has morenegative charges than positive charges in an aqueous medium with aphysiological pH (e.g., pH7.4), or a cationic lipid, which has morepositive charges than negative charges in the aqueous medium. Lipidswhich have both cationic and anionic groups (i.e., so-called amphotericlipids) should also be judged based on the same criterion.

The charged lipid 3 retains the drug 4 within the pharmaceuticalcomposition 1 via electrostatic bonding. The charged lipid 3 may onlyhave an electrical charge opposite to the charge of the drug 4 at leastin the storage environment of the pharmaceutical composition 1. Thecharged lipid 3 should preferably have a charge opposite to that of thedrug 4 under physiological environments, such as in blood (e.g., pH7.4).

The charged lipids 3 are being attracted to the hydrophobicpolymer-chain segments 2 b by the following mechanism. The carriercomposition, which is a base material for the pharmaceutical composition1 of the present invention, can be formed by a method including, e.g.,the step of suspending the block copolymer units 2 and the chargedlipids 3 into an aqueous solution. The hydrophobic polymer-chainsegments 2 b of the block copolymer units 2 cannot disperse, but form anaggregate, in the aqueous solution due to their hydrophobicity, whilethe hydrophilic polymer-chain segments 2 a can disperse, and movefreely, in the aqueous solution. Thus, the block copolymer units 2 arearranged radially in the aqueous solution, with the hydrophobicpolymer-chain segments 2 b radially inside and the hydrophilicpolymer-chain segments 2 a radially outside. The charged lipids 3 arebeing attracted to the hydrophobic polymer-chain segments 2 b, sincethey are highly hydrophobic and have higher affinity for the hydrophobicpolymer-chain segments 2 b than for water or the hydrophilicpolymer-chain segments 2 a. Thus, the charged lipids 3 are arranged awayfrom the outer surface of the carrier composition and, even after thefreezing operation explained below, are kept being attracted to thehydrophobic polymer-chain segments 2 b.

In the pharmaceutical composition 1 of the present invention, thecharged lipids 3 are being attracted to the hydrophobic polymer-chainsegments 2 b, whereby the outer surface of the pharmaceuticalcomposition 1 is prevented from being charged so as to attract asubstance which has a charge opposite to that of the charged lipids 3(e.g., blood proteins). This state can be confirmed based on, i.e.,whether the absolute value of the zeta potential of the pharmaceuticalcomposition 1 is lower than a predetermined value. More specifically,the absolute value of the zeta potential of the pharmaceuticalcomposition 1 should preferably be 15 mV or lower, more preferably 12 mVor lower, still more preferably 6 mV or lower, even more preferably 3 mVor lower. The zeta potential can be measured by adding the carriercomposition or the pharmaceutical composition 1 to 10 mM HEPES buffersolution (pH 7.4) in such an amount as for the ratio of the totalcharged lipids to the buffer solution to be 0.1 mg/ml.

The ratio by weight of the amount of the block copolymer units to theamount of the charged lipids 3 should preferably be 1.0 or higher, morepreferably 1.5 or higher, still more preferably 2.0 or higher, andpreferably 50 or lower, more preferably 20 or lower, still morepreferably 10 or lower. The higher the ratio, the lower the absolutevalue of the zeta potential of the pharmaceutical composition 1. On theother hand, drugs can be introduced more actively into the particle asthe ratio of the charged lipids 3 becomes higher, for which reason theratio should preferably be limited to 50 or lower as mentioned above.

The lipids may be a simple lipid, a conjugated lipid or a derived lipid.Examples thereof include phospholipids, glycoglycerolipids,glucosphingolipids, sphingoids and sterols. Specifically, examples ofcationic lipids include 1,2-dioleoyl-3-trimethylammoniopropane (DOTAP),N-(2,3-dioleoyloxypropan-1-yl)-N,N,N-trimethylammonium chloride (DOTMA),2,3-dioleoyloxy-N-[2-(sperminecarboxyamide)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE),1,2-dioleoyloxypropyl-3-diethylhydroxyethylammonium bromide (DORIE), and3β-[N—(N′N′-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol). Examplesof anionic lipids include cardiolipin, diacylphosphatidylserine,diacylphosphatidic acid, N-succinyl phosphatidylethanolamine (N-succinylPE), phosphatidic acid, phosphatidylinositol, phosphatidylglycerol,phosphatidylethylene glycol, and cholesterol succinate. Thepharmaceutical composition 1 may contain two or more kinds of chargedlipids 3.

The hydrophilic polymer-chain segment 2 a should preferably be awater-soluble polymer-chain segment made of polyethyleneglycol orpolyoxyethylene. The molecular weight of the hydrophilic polymer-chainsegment 2 a should preferably be 2,500 Da or higher, more preferably5,000 Da or higher, still more preferably 8,000 Da or higher, andpreferably 200,000 Da or lower, more preferably 20,000 Da or lower,still more preferably 15,000 Da or lower. The hydrophobic polymer-chainsegment 2 b should preferably be a segment derived from a polyamino acidchain, part or all of which can form the α-helix in the pharmaceuticalcomposition 1, whereby the charged lipids 3 can be attracted to theα-helix of the polyamino acid chain, i.e., dispersed around the α-helix.The number of repeating units in the hydrophobic polymer-chain segment 2b should preferably be 10 or higher, more preferably 20 or higher, andpreferably 200 or lower, more preferably 100 or lower, still morepreferably 60 or lower. In order to reduce the absolute value of thezeta potential of the pharmaceutical composition 1, i.e., to reduce thesurface charge of the pharmaceutical composition 1 (to be closer toneutral), the size of the hydrophilic polymer-chain segment 2 a(molecular weight) should preferably be larger than the size of thehydrophobic polymer-chain segment 2 b (the number of repeating units) inthe block copolymer unit 2. The hydrophilic polymer-chain segment 2 aand/or the hydrophobic polymer-chain segment 2 b may form a branchedstructure. For example, a single chain of one segment may be coupled totwo or more chains of the other segment.

The hydrophilic polymer-chain segment 2 a and the hydrophobicpolymer-chain segment 2 b may also have a charged substituent such as anamino group and carboxy group, as long as the outer particle surface ofthe pharmaceutical composition 1 does not bear a charge which canattract a charged substance.

The hydrophilic polymer-chain segment 2 a and the hydrophobicpolymer-chain segment 2 b can be linked to each other by covalentlybonding the termini of their main chains. More specifically, examples ofthe block copolymer unit 2 are the compounds represented by generalformulae (I) and (II). The pharmaceutical composition 1 may contain twoor more kinds of the block copolymer units 2.

In formulae (I) and (II),

R¹ and R³, independently of each other, is either hydrogen atom or agroup represented by R⁸(R⁹)CH(CH₂)_(q)— (where R⁸ and R⁹: i)independently of each other, is hydrogen atom, C₁₋₆ alkoxy group,aryloxy group, aryl-C₁₋₃-oxy group, cyano group, carboxy group, aminogroup, C₁₋₆-alkoxy carbonyl group, C₂₋₇-acylamide group, tri-C₁₋₆-alkylsiloxy group, siloxy group, or silylamino group; ii) together with eachother, form ethylene dioxy group or propylene dioxy group, which areeither unsubstituted or substituted with C₁₋₃-alkyl group; or iii)together with CH group to which they are bound, form formyl group);

q is an integer of from 0 to 10;

R² is hydrogen atom, saturated or unsaturated C₁-C₂₉ aliphatic carbonylgroup, or arylcarbonyl group;

R⁴ is hydroxy group, saturated or unsaturated C₁-C₃₀-aliphatic oxygroup, or aryl-lower-alkyloxy group;

R⁵ is —O— or —NH—;

R⁶ is hydrogen atom, phenyl group, benzyl group, —(CH₂)₄-phenyl group,C₄-C₁₆ alkyl group which is either unsubstituted or substituted withamino group or carbonyl group, or sterol derivative residue;

R⁷ is methylene group;

n is an integer of from 55 to 4,600;

x is an integer of from 10 to 200;

m is an integer of from 0 to 200 (wherein when m is one or more, the(COCHNH) units and the (COR⁷CHNH) unit(s) can be arranged in any order,and when m is two or more, R⁶ is selected for each amino acid unitindependently of each other and can be arranged in the block copolymerin a random order, provided that hydrogen atoms must not account for 75%or more of R⁶);

y is 1 or 2;

L¹ is a linking group selected from —NH—, —O—, —O—Z—NH—, —CO—, —CH₂—,and —O—Z—S—Z—NH— (where Z, independently of each other, means C₁-C₆alkylene group); and

L² is a linking group selected from —OCO—Z—CO—, and —NHCO—Z—CO— (whereinZ is C₁-C₆ alkylene group).

In formulae (I) and (II),

n is an integer of preferably 110 or larger, more preferably 180 orlarger, and preferably 460 or smaller, more preferably 340 or smaller;

x is an integer of preferably 20 or larger, and preferably 100 orsmaller, more preferably 60 or smaller; and

m is an integer of preferably 100 or smaller, more preferably 60 orsmaller.

The block copolymer unit 2 may be an anionic polymer, a cationicpolymer, or a neutral polymer. As used herein, polymers which have morenegative charges than positive charges in an aqueous medium with aphysiological pH (e.g., pH7.4) are regarded as anionic, polymers whichhave more positive charges than negative charges in the aqueous mediumare regarded as cationic, and polymers which have substantially equalamounts of positive charges and negative charges in the aqueous mediumare regarded as neutral.

The block copolymer unit 2 can be formed, e.g., by coupling a polymerhaving a hydrophilic polymer chain and a polymer having a polyamino acidchain in a know manner, optionally after purifying the polymers, ifnecessary, to restrict the molecular weight distribution. The blockcopolymer unit 2 according to formula (I) also can be formed, e.g., bythe steps of: performing anion living polymerization using an initiatorwhich can add R¹ to form a polyethyleneglycol chain; introducing anamino group to the growing end; and polymerizing, at the amino end, anN-carboxy anhydride (NCA) of a protected amino acid, such asNε-Z-L-lysin, β-benzyl-L-aspartate, or γ-benzyl-L-glutamate.

The carrier composition can be formed, e.g., as follows. First, a blockcopolymer unit and a charged lipid, optionally together with a neutrallipid, are fully dissolved or dispersed into a forming solutioncontaining an organic solvent, after which the organic solvent isremoved by evaporation. Examples of organic solvents include acetone,dichloromethane, dimethylformamide, dimethylsulfoxide, acetonitrile,tetrahydrofuran, and methanol. The forming solution may contain two ormore organic solvents, and also may contain a small amount of water. Theresultant solid or paste is combined with water or an aqueous solutioncontaining an additive such as an appropriate salt or stabilizer,followed by stirring to disperse the block copolymer unit and thelipid(s). The resultant product is further dispersed/pulverized by meansof, e.g., ultrasonic irradiation, high-pressure emulsification orextruder to thereby form the carrier composition.

The drug 4 is retained in the pharmaceutical composition 1 viaelectrostatic bonding with the charged lipid 3. Thus, the link betweenthe charged lipid 3 and the drug 4 is reversible, and does not involveany chemical structural change. The drug 4 can be introduced into thecarrier composition either by adding the drug 4 to the forming solutionin the production of the carrier composition, or by adding the carriercomposition to a solution of the drug 4.

Examples of the drug 4 include: anionic compounds, which have morenegative charges than positive charges in an aqueous medium with aphysiological pH (e.g., pH7.4); and cationic compounds, which have morepositive charges than negative charges in the aqueous medium. Thecompounds should preferably be macromolecular compounds.

The drug 4 should preferably be a biomacromolecule. The biomacromoleculeherein means a macromolecule of biological origin or a structuralanalogue thereto, and more specifically, should preferably be at leastone selected from a protein and a nucleic acid. There are no limitationsto the alternatives and sizes of proteins and nucleic acids, and theproteins include peptides. Such a biomacromolecule is at least partiallyhydrophilic; especially, nucleic acids exhibit very high hydrophilicity.

Accordingly, even if preparing a composite of a biomacromolecule (e.g.,a nucleic acid) and a lipid charged oppositely to the biomacromolecule,and trying to introduce the composite into a conventional polymermicelle particle which does not contain a charged lipid, it would bedifficult to transfer the composite to inside the polymer micelleparticle by mean of hydrophobic interaction. This is because thebiomacromolecule having a polar portion would surround the charged lipidand render the composite surface nearly hydrophilic (i.e., much lesshydrophobic than at least than the hydrophobic portion of the blockcopolymer existing near the polymer micelle surface).

In order to prevent the drug 4 either from disengaging from thepharmaceutical composition 1 in blood too early or from beingencapsulated in the pharmaceutical composition 1 for too long a time,the charge ratio between the charged lipid 3 and the drug 4 in thepharmaceutical composition 1 should preferably be controlled to bewithin a particular range. When the drug 4 is, e.g., a nucleic acid, thecharge ratio can be defined as [the mol concentration of cationic groupsof the charged lipid contained in the pharmaceutical composition]/[themol concentration of phosphoric groups in the nucleic acid]. On theother hand, when the drug 4 is a compound which has both anionic andcationic groups, e.g., a protein, the charge ratio can be defined as[the mol concentration of cationic groups of the charged lipid containedin the pharmaceutical composition]/([the mol concentration of groups inthe drug which are charged oppositely to the charged lipid]—[the molconcentration of groups in the drug which are charged similarly to thecharged lipid]). The charge ratio should preferably be 0.5 or higher,more preferably one or higher, still more preferably 2 or higher, andpreferably 50 or lower, more preferably 20 or lower, still morepreferably 10 or lower.

The average particle sizes of the carrier composition and thepharmaceutical composition 1 should preferably be 10 nm or larger, morepreferably 30 nm or larger, and preferably 300 nm or smaller, morepreferably 200 nm or smaller.

EXAMPLES

The present invention will be explained in more detail below byreferring to Examples.

I. Preparation of Particulate Pharmaceutical Compositions:

Particulate pharmaceutical compositions were prepared in accordance withthe following procedure, and subjected to the measurements explainedbelow.

I-1. Preparation of Particulate Carrier Compositions:

I-1-1. Preparation of Particulate Carrier Composition a (with PEG-PBLG):

Five grams of α-methoxy-ω-amino-polyethyleneglycol (hereinafter alsoreferred to as “PEG”) having a weight-average molecular weight (Mw) of10000 (Manufactured by NOF Corp.) was dissolved into 50 ml of dimethylsulfoxide, which was reacted with 5.5 g (42 parts with respect topolyethyleneglycol) of N-carboxy anhydride (NCA) of γ-benzyl-L-glutamate(hereinafter also referred to as “PBLG”) at 40° C. for 24 hours. Thereaction solution was dropped into 1 L of a mixture solvent of hexaneand ethyl acetate (volume ratio 1:1) to cause precipitation of apolymer, which was recovered by filtration under reduced pressure andthen dried to yield 8.6 g of a solid product. This product was dissolvedinto 86 ml of DMF, with which 432 μl of acetic anhydride was mixed andreacted at 40° C. for 24 hours. The reaction solution was dropped into 1L of a mixture solvent of hexane and ethyl acetate (volume ratio 1:1) tocause precipitation of a polymer, which was recovered by filtrationunder reduced pressure and then further dried to yield 8.1 g ofpolyethyleneglycol-poly(γ-benzyl-L-glutamate)-Ac block copolymer(hereinafter also referred to as “PEG-PBLG”), which is a neutralpolymer. The structural formula of PEG-PBLG is shown below. ¹H-NMRanalysis revealed that the degree of polymerization of the PBLG blockwas 40.

The resultant PEG-PBLG(10-40) (block copolymer unit) was mixed withDOTAP (cationic lipid) and DOPE (neutral lipid) at the ratios of 2.5/1/1by weight in chloroform, and dried under reduced pressure until solidwas obtained. This mixture was combined with 10 mM HEPES buffer (pH7.4),stirred at 4° C. overnight, pulverized by ultrasonic irradiation, andpassed through a 0.22 μm filter to thereby yield a solution of lipidmicelle containing PEG-PBLG (hereinafter also referred to as“particulate carrier composition A”).

I-1-2. Preparation of Particulate Carrier Composition B (withPEG-pGlu(Bn)):

PEG-PBLG was alkali-treated to deprotect the benzyl groups of theglutamic acid side chains, whereby polyethyleneglycol/poly(L-glutamicacid) block copolymer was obtained (hereinafter also referred to as“PEG-pGlu”). The glutamic acid side chains of PEG-pGlu were partiallymodified with benzyl groups (PhCH₂) via condensation reaction usingbenzyl alcohol to thereby yield polyethyleneglycol/benzyl-introducedpoly(L-glutamic acid) block copolymer (hereinafter also referred to as“PEG-pGlu(Bn)”), which is an anionic polymer. ¹H-NMR analysis revealedthat the number of benzyl groups introduced was 35 per polymer. Thestructural formula of PEG-pGlu(Bn) is shown below.

One milliliter of an acetone solution (50 mg/mL) of the resultantPEG-pGlu(Bn) (block copolymer unit) was mixed with 0.5 mL of a methanolsolution (40 mg/mL) of DOTAP (cationic charged lipid) and 0.5 mL of amethanol solution (40 mg/mL) of DOPE (neutral lipid), and dried underreduced pressure until solid was obtained. The resultant mixture wascombined with 2.5 mL of 100 mM sodium phosphate buffer (pH7.4), stirredfor three hours at room temperature, pulverized by ultrasonicirradiation (130 W, 1 second pulse, 10 minutes), and passed through a0.22 μm filter (Millex GP, Millipore) to thereby yield a solution oflipid micelle containing PEG-pGlu(Bn) (hereinafter also referred to as“particulate carrier composition B”).

I-2. Preparation of Pharmaceutical Compositions Via Freezing Operation:

I-2-1. Preparation of Pharmaceutical Compositions UsingFreezing-and-Thawing Operation:

SiRNA was dissolved into 10 mM HEPES buffer (pH7.4) to prepare 40 μlsiRNA solution, which was mixed with the lipid micelle solutioncontaining PEG-PBLG as a block copolymer unit (particulate carriercomposition A), 10 mM HEPES buffer and 50% sucrose solution to prepare asolution of lipid micelle containing, as a drug, siRNA shown in eachExample (hereinafter also referred to as “particulate pharmaceuticalcomposition A”). The concentrations of siRNA and sucrose in theparticulate pharmaceutical composition solution were adjusted to 10 μMand 10%, respectively. The mixture ratio between the cationic lipid(DOTAP) and the siRNA was also adjusted so as for the charge ratio (+/−)of the concentration of positive charges (cationic group) of thecationic lipid (DOTAP) to the concentration of negative charges(phosphoric group) of siRNA to be 8.

In each of the Examples described in the present description, siRNA wasselected from the ones explained below, all of which are available fromNippon EGT Co., Ltd.

-   -   siRNA(Luc): Designed to target the firefly luciferase gene, this        siRNA is composed of a sense strand of        5′-CUUACGCUGAGUACUUCGAdTdT-3′ (SEQ ID NO:1) and an antisense        strand of 5′-UCGAAGUACUCAGCGUAAGdTdT-3′ (SEQ ID NO:2)        double-stranded in a conventional manner.    -   siRNA(Plk1): Designed to target the human Plk1 (Polo-like        kinase 1) gene, this siRNA is composed of a sense strand of        5′-CCAUUAACGAGCUGCUUAAdTdT-3′ (SEQ ID NO:3) and an antisense        strand of 5′-UUAAGCAGCUCGUUAAUGGdTdT-3′ (SEQ ID NO:4)        double-stranded in a conventional manner. The Plk1 gene is a        kinase which plays a role in the M phase of cell division.        SiRNA(Plk1) induces apoptosis when introduced into the cell.    -   F-siRNA(Luc): This siRNA is formed in the same manner as        siRNA(Luc) except that the antisense strand of SEQ ID NO:2 is        Cy3-labeled at the 5′ end (5′-Cy3-UCGAAGUACUCAGCGUAAGdTdT-3′).

The resultant solution of siRNA-encapsulated lipid micelle (particulatepharmaceutical composition A) was subjected to the measurementsexplained below, either without any other treatment or after receivingfreezing-and-thawing operation once, twice, or thrice. Eachfreezing-and-thawing operation was carried out by freezing thecomposition at −80° C. for 12 hours, and thawing it at room temperaturefor an hour.

Hereinafter, the untreated particulate pharmaceutical composition A isalso referred to as “the pharmaceutical composition of Reference Example1,” the particulate pharmaceutical composition A which received thefreezing-and-thawing operation once is also referred to as “thepharmaceutical composition of Example 1,” the particulate pharmaceuticalcomposition A which received the freezing-and-thawing operation twice isalso referred to as “the pharmaceutical composition of Example 2,” andthe particulate pharmaceutical composition A which received thefreezing-and-thawing operation thrice is also referred to as “thepharmaceutical composition of Example 3.” In addition, each of thepharmaceutical compositions of Reference Example 1 and Examples 1 to 3,as well as the pharmaceutical compositions of Reference Example 2 andExample 4 explained below, is also referred to simply as “sample”without distinction.

In each of the pharmaceutical compositions of Reference Example 1 andExamples 1 to 3, the charged lipid (DOTAP) retains the drug (siRNA) inthe particle via electrostatic bonding while being attracted to thehydrophobic polymer-chain segment, whereby the outer particle surface isprevented from bearing a charge which can attract a substance chargedoppositely to the charged lipid. In addition, in each of thepharmaceutical compositions of Reference Example 1 and Examples 1 to 3,the hydrophobic polymer-chain segment derived from a polyamino acidchain (PBLG) is believed to form the α-helix, around which the chargedlipid (DOTAP) is dispersed. Further, in each of the pharmaceuticalcompositions of Examples 1 to 3, the drug is positioned inside thehydrophobic polymer-chain segment.

I-2-2. Preparation of the Pharmaceutical Composition Using Freeze-DryingOperation:

To 100 μM siRNA aqueous solution, a solution of lipid micelle containingPEG-pGlu(Bn) as the block copolymer unit (particulate carriercomposition B) and sucrose were mixed, and let stand still at 4° C. fortwo hours to prepare a solution of lipid micelle containing, as a drug,siRNA shown in each Example (hereinafter also referred to as“particulate pharmaceutical composition B”). The concentrations of siRNAand sucrose in the particulate pharmaceutical composition solution wereadjusted to 20 μM and 10%, respectively. The mixture ratio between thecationic lipid (DOTAP) and the siRNA was also adjusted so as for thecharge ratio (+/−) of the concentration of positive charges (cationicgroup) of the cationic lipid (DOTAP) to the concentration of negativecharges (phosphoric group) of siRNA to be 8.

The resultant solution of siRNA-encapsulated lipid micelle (particulatepharmaceutical composition B) was subjected to the measurementsexplained below, either without any other treatment or after it wasreceived freeze-drying operation in a conventional manner to form astock, and then dissolved into water again. The freeze-drying operationwas carried out using Triomaster II A-04 (manufactured by NISSEI Ltd.).

Hereinafter, the untreated particulate pharmaceutical composition B isalso referred to as “the pharmaceutical composition of Reference Example2,” and the particulate pharmaceutical composition B which received thefreeze-drying operation is also referred to as “the pharmaceuticalcomposition of Example 4.”

In each of the pharmaceutical compositions of Reference Example 2 andExample 4, the charged lipid (DOTAP) retains the drug (siRNA) in theparticle via electrostatic bonding while being attracted to thehydrophobic polymer-chain segment, whereby the outer surface of theparticulate composition is prevented from gathering a charged substance,which has an opposite charge of the charged lipid, as a corollary ofless electrical charge. In addition, in each of the pharmaceuticalcompositions of Reference Example 2 and Example 4, the hydrophobicpolymer-chain segment derived from a polyamino acid chain (pGlu(Bn)) isbelieved to form the α-helix, around which the charged lipid (DOTAP) isdispersed. Further, in the pharmaceutical composition of Example 4, thedrug is positioned radially inside the hydrophobic polymer-chainsegment.

II. Measurements of Particulate Pharmaceutical Compositions:

II-1. Light-Scattering Measurement:

This measurement was carried out using, as a sample, each of thepharmaceutical compositions of Reference Example 1 (untreated), Example1 (freezing-and-thawing once), Example 2 (freezing-and-thawing twice)and Example 3 (freezing-and-thawing thrice), which were prepared inaccordance with the procedure described in “I. Preparation ofparticulate pharmaceutical compositions” using siRNA(Luc) as the siRNAand PEG-PBLG (neutral polymer) as the block copolymer unit, as well asReference Example 2 (untreated) and Example 4 (freeze-drying), whichwere prepared likewise but using PEG-pGlu(Bn) (cationic polymer) as theblock copolymer unit.

Each sample was diluted with 10 mM HEPES buffer so as for the siRNAconcentration to be 1 μM. The micelles in each sample were measured forparticle size, scattering intensity, and the absolute value of the zetapotential with the light-scattering analyzer (Zetasizer Nano ZS, MalvernInstruments).

The results of measurement for particle size and zeta potential areshown in Table 1.

Comparison of the pharmaceutical compositions of Examples 1 to 3 withthe pharmaceutical composition of Reference Example 1, each of which wasprepared using PEG-PBLG (neutral polymer) as the block copolymer unit,shows that in each of the pharmaceutical compositions of Example 1(freezing-and-thawing once), Example 2 (freezing-and-thawing twice) andExample 3 (freezing-and-thawing thrice), there were no significantchanges in the particle size and the scattering intensity of themicelles, but there was an increase in the absolute value of the zetapotential, compared to the pharmaceutical composition of ReferenceExample 1 (untreated). Taking into consideration the fact that theparticle size and the scattering intensity did not change substantially,the increase in zeta potential is believed to be due to relativeincrease in the positive charge at the micelle surface caused bytransfer of siRNA into the inner part of the micelle, not due todissociation of the lipid from the micelle.

TABLE 1 Particle Absolute value of Pharmaceutical composition size (nm)zeta potential (mV) Reference Example 1 116 6.85 (PEG-PBLG, untreated)Example 1 120 9.00 (PEG-PBLG, freezing/thawing × 1) Example 2 122 11.10(PEG-PBLG, freezing/thawing × 2) Example 3 122 11.10 (PEG-PBLG,freezing/thawing × 3) Reference Example 2 147 0.455 (PEG-pGlu(Bn),untreated) Example 4 190 0.421 (PEG-pGlu(Bn), freeze-drying)II-2. Measurement of Encapsulation Rate:

This measurement was carried out using, as a sample, each of thepharmaceutical compositions of Reference Example 1 (untreated), Example1 (freezing-and-thawing once), Example 2 (freezing-and-thawing twice)and Example 3 (freezing-and-thawing thrice), which were prepared inaccordance with the procedure described in “I. Preparation ofparticulate pharmaceutical compositions” using siRNA(Luc) as the siRNAand PEG-PBLG (neutral polymer) as the block copolymer unit, as well asReference Example 2 (untreated) and Example 4 (freeze-drying), whichwere prepared likewise but using PEG-pGlu(Bn) (cationic polymer) as theblock copolymer unit.

II-2-1. Preparation of Calibration Curve:

A series of diluted reference solutions, each of which has a volume of50 μL, was prepared by diluting 40 μM siRNA solution with 10 mM HEPESbuffer serially by one third (resulting in 11 reference solutionsranging from 40 μM to 0.68 nM). Each reference solution of 50 μL fromthe resultant series was measured for fluorescence intensity to preparea calibration curve in accordance with the following procedure. 50 μL ofeach reference solution was mixed with 750 μL of 10 mM HEPES buffer and200 μL of 1% TRITONX-100 aqueous solution. 100 μL from the resultantmixture solution was put into each well of a 96-well plate (black).After adding 100 μL of PicoGreen™ solution (diluted by 1/200 with 10 mMHEPES buffer) to each well followed by mixing, the mixture was leftstanding at room temperature in the absence of light for five minutes,and measured for fluorescence intensity using a plate reader to plot acalibration curve. Since the resultant calibration curve was linearwithin the siRNA concentration range of from 1.48 μM to 2 nM, thesamples were measured within this range.

II-2-2. Measurement of Samples:

Each sample was diluted (standardized) with 10 mM HEPES buffer (pH7.4)so as for the siRNA concentration to be 1 μM. 500 μL of the solution wasultracentrifuged (100,000×g, 4° C., an hour) to sediment the micelles,and 50 μL of the supernatant was collected stilly. The collectedsupernatant was measured for fluorescence intensity in accordance withthe procedure explained in “II-2-1. Preparation of calibration curve,”and the obtained fluorescence intensity was collated with thecalibration curve to determine the standardized non-encapsulated siRNAconcentration, which is the concentration of siRNA in the supernatantderived from each sample. The siRNA encapsulation rate of the micellesin each sample was determined in accordance with the following equation:SiRNA encapsulation rate (%)=(1−x)×100where x means the standardized non-encapsulated siRNA concentration(μM).II-2-3. Results:

The resultant siRNA encapsulation rates are shown in Table 2. In each ofthe pharmaceutical compositions of Reference Examples 1 and 2 andExamples 1 to 4, the supernatant after ultracentrifuging contained verylittle siRNA, and almost all of the siRNA was included in the micelles.

TABLE 2 Encapsulation rate Pharmaceutical composition (%) ReferenceExample 1 99.1 (PEG-PBLG, untreated) Example 1 99.5 (PEG-PBLG,freezing/thawing × 1) Example 2 99.5 (PEG-PBLG, freezing/thawing × 2)Example 3 99.4 (PEG-PBLG, freezing/thawing × 3) Reference Example 2 96.6(PEG-pGlu(Bn), untreated) Example 4 98.3 (PEG-pGlu(Bn), freeze-drying)II-3. Evaluation of Encapsulation Stability:

This measurement was carried out using, as a sample, each of thepharmaceutical compositions of Reference Example 1 (untreated) andExample 3 (freezing-and-thawing thrice), which were prepared inaccordance with the procedure described in “I. Preparation ofparticulate pharmaceutical compositions” using siRNA(Luc) as the siRNAand PEG-PBLG (neutral polymer) as the block copolymer unit, as well asReference Example 2 (untreated) and Example 4 (freeze-drying), whichwere prepared likewise but using PEG-pGlu(Bn) (cationic polymer) as theblock copolymer unit.

Dextran sulfate, an anionic macromolecular, was added to each sample,and the ratio of the siRNA replaced with dextran sulfate and releasedfrom the micelles (siRNA release rate) was measured to evaluate theencapsulation stability of each sample. More specifically, theevaluation was carried out as follows. Each sample was mixed with anexcess dextran sulfate (the charge ratio of which to siRNA is 80), anddiluted with 10 mM HEPES buffer (pH7.4) so as for the siRNAconcentration to be 1 μM (i.e., standardized). 500 μL of the obtainedsolution was ultracentrifuged (100,000×g, 4° C., one hour) to sedimentthe micelles, and 50 μL of the supernatant was collected stilly. Thecollected supernatant was measured for fluorescence intensity inaccordance with the procedure explained in “II-2-1. Preparation ofcalibration curve,” and the obtained fluorescence intensity was collatedwith the calibration curve obtained in “II-2-2. Measurement of samples”to determine the standardized non-encapsulated siRNA concentration inthe supernatant derived from each sample, which indicates the amount ofthe siRNA released from the micelles in each sample. The siRNA releaserate (the ratio of the amount of siRNA replaced with dextran sulfate andreleased from the micelles to the amount of siRNA originally included inthe micelle) was determined in accordance with the following equation:siRNA release rate (%)={(y−x)/1}{y−x}×100where x means the standardized non-encapsulated siRNA concentration(μM); and y means the amount of siRNA released from the micelles (μM).

The results are shown in FIG. 3. Comparison of the pharmaceuticalcompositions of Example 3 and Reference Example 1, both of which wereprepared using PEG-PBLG (neutral polymer) as the block copolymer unit,shows that the pharmaceutical composition of Example 3, which wasobtained through freezing operation, had a reduced siRNA release rate,which is about half that of the pharmaceutical composition of ReferenceExample 1, which received no freezing operation. Comparison of thepharmaceutical compositions of Example 4 and Reference Example 2, bothof which were prepared using PEG-pGlu(Bn) (cationic polymer) as theblock copolymer unit, also shows that the pharmaceutical composition ofExample 4, which was obtained through freezing operation, had a reducedsiRNA release rate which is about 30% as that of the pharmaceuticalcomposition of Reference Example 2.

II-4. Evaluation of Remaining Rate in Blood:

This measurement was carried out using, as a sample, each of thepharmaceutical compositions of Reference Example 1 (untreated) andExample 3 (freezing-and-thawing thrice), which were prepared inaccordance with the procedure described in “I. Preparation ofparticulate pharmaceutical compositions” using F-siRNA(Luc) as the siRNAand PEG-PBLG (neutral polymer) as the block copolymer unit, as well asReference Example 2 (untreated) and Example 4 (freeze-drying), whichwere prepared likewise but using PEG-pGlu(Bn) (cationic polymer) as theblock copolymer unit.

Each sample was administered to Balb/c mice (obtained from Charles RiverLaboratories Japan, Inc.) via the tail vein, and 200 μl of blood wascollected via the inferior vena cava one hour after. The dosage of eachsample for each mouse was determined so as for the ratio of F-siRNA tothe weight of the mouse to be 1 mg/kg. The collected blood wascentrifuged with 2000×g at 4° C. for 10 minutes, and 80 μl of plasma wascollected from the supernatant. The plasma was measured for fluorescenceintensity using a plate reader (POWERSCAN HT, manufactured by DainipponSumitomo Pharma Co., Ltd.) (excitation wavelength: 485 nm; fluorescencewavelength: 528 nm) to determine the quantity of F-siRNA circulating inblood, as an indicator of remaining rate in blood.

The results are shown in FIG. 4. Comparison of the pharmaceuticalcompositions of Example 3 and Reference Example 1, both of which wereprepared using PEG-PBLG (neutral polymer) as the block copolymer unit,shows that the pharmaceutical composition of Example 3, which wasobtained through freezing operation, resulted in more F-siRNA remainingin blood, i.e., higher remaining rate in blood. Comparison of thepharmaceutical compositions of Example 4 and Reference Example 2, bothof which were prepared using PEG-pGlu(Bn) (cationic polymer) as theblock copolymer unit, also shows that the pharmaceutical composition ofExample 4, which was obtained through freezing operation, resulted inmore F-siRNA remaining in blood, higher remaining rate in blood.

The invention claimed is:
 1. A particulate pharmaceutical compositioncomprising: a plurality of block copolymer units, each unit having ahydrophobic polymer-chain segment and a hydrophilic polymer-chainsegment, the hydrophobic polymer-chain segment being a polyamino acidsegment, the plurality of block copolymer units being arranged radiallywith the hydrophobic polymer-chain segments radially inside and thehydrophilic polymer-chain segments radially outside; a plurality ofcharged lipids carrying a first charge, the plurality of charged lipidsbeing attracted to the hydrophobic polymer-chain segment; and a drugcarrying a second charge opposite to the first charge and comprising abiomacromolecule selected from the group consisting of proteins andnucleic acids, wherein the particulate pharmaceutical composition issubjected to freezing, wherein the drug is positioned radially insiderelative to the hydrophobic polymer-chain segments as a result of thefreezing such that the drug is prevented from disengaging from theparticulate pharmaceutical composition, and wherein the particulatepharmaceutical composition has an absolute zeta potential that is higherthan that of a particulate pharmaceutical composition that has not beensubjected to the freezing.
 2. The particulate pharmaceutical compositionaccording to claim 1, wherein part or all of the polyamino acid chainforms an α-helix.
 3. The particulate pharmaceutical compositionaccording to claim 2, wherein the charged lipid is dispersed around theα-helix.